1. Field of the Invention
The present invention relates to a novel method of differential assay for .alpha.-amylase isozymes and a kit for the same. More particularly, the present invention relates to a novel method of differential assay for human pancreatic .alpha.-amylase and human salivary .alpha.-amylase and a kit used for such a method. According to the method for measurement and the kit of the present invention, marked effects are achieved that differential assay for human pancreatic .alpha.-amylase and human salivary .alpha.-amylase can be extremely easily performed with good accuracy using a spectrophotometer, a fluorophotometer, etc. widely applied to the field of clinical examination and the method is readily applicable to an automated analyzer. The measurement method and kit of the present invention are thus extremely useful for daily clinical tests such as analysis of hyperamylasemia research for disease, etc.
2. Description of the Related Art
It is important for medical diagnosis to measure .alpha.-amylase activity in a vital sample, in particular, in human saliva, pancreatic juice, blood or urine. For example, in pancreatic disease, pancreatic cancer and parotitis, the .alpha.-amylase activity in blood or urine shows a marked rise as compared to the normal value.
In addition, for purposes of analyzing hyperamylasemia or research for disease, it is important to determine the .alpha.-amylase activity in blood, dividing into its isozyme, and such has been applied to daily clinical tests.
There are hitherto known various methods for separation of human pancreatic .alpha.-amylase and human salivary .alpha.-amylase, including (1) separation utilizing difference in charge [Clin. Chim. Acta, 54,137, (1974)], (2) gel filtration [Clin. Chem., 18, 1493, (1972)], (3) a method utilizing affinity chromatography, (4) an immunological method [J. Biochemistry, 97, 1357, (1985)], (5) a method using an .alpha.-amylase inhibitor [Clin. Chem., 23, 560, (1977)], and the like.
Among them, methods that are currently applicable to clinical tests are (1) the method in which the separation is effected by electrophoresis utilizing difference in charge [Clinical Pathology, "An Analysis of Isoenzyme and its Significance", a Special Number 43, p. 17 (1981)] and (5) the method using an amylase inhibitor which has been frequently adopted recently.
Electrophoretic methods suited for clinical tests include a method using a cellulose acetate membrane or thin layer polyacrylamide gel, or the like but any of them involves defects that operations for the measurement are complicated and many hours is required for the measurement.
On the other hand, the method using the .alpha.-amylase inhibitor comprises, utilizing more potent inhibitory action of a wheat-derived amylase inhibitor on salivary .alpha.-amylase than on pancreatic .alpha.-amylase, determining the ratio of both amylases. At present, any inhibitor that specifically inhibits completely either pancreatic .alpha.-amylase or salivary .alpha.-amylase has not been found. In such a method, therefore, the ratio of pancreatic .alpha.-amylase activity to salivery .alpha.-amylase activity in a sample is read out from a calibration curve prepared using standard enzyme solutions of known concentrations. Such a method has been recently widely used because the measurement can be made relatively simply.
In order to determine the ratio of pancreatic .alpha.-amylase activity to salivary .alpha.-amylase activity according to this method, however, it is necessary to measure twice, i.e., in the case of using the inhibitor and in the case of using no inhibitor. Accordingly, the method still involves complicated operations.
On the other hand, the present inventors have already found out that the the actions of the isozymes on oligosaccharide derivatives differs and that the ratio of products produced therefrom also differs depending upon the isozymes and filed a patent application directed to a method utilizing those findings for differential assay of .alpha.-amylase isozymes (European Patent Publication No. 104047). This method is concerned with differential assay of human pancreatic .alpha.-amylase and human salivary .alpha.-amylase which comprises using as a substrate oligosaccharide having a certain modifying group, namely, a modifying group having a fluorescent property or a modifying group which absorbs UV, and measuring degradation products formed upon hydrolysis with an .alpha.-amylase by high performance liquid chromatography. According to this method, the differential assay of .alpha.-amylase isozymes has become much easier than in the prior arts. In this method, however, special equipments such as high performance liquid chromatography, etc. must be used; in addition, the method has some problem such as time-consuming, and is not sufficiently satisfactory yet. It has thus been desired to develop a method for differential assay of .alpha.-amylase isozymes applicable to spectrophotometry, fluorophotometry, automated analyzers, etc. widely spread in the field of clinical tests and having excellent handling ability of samples.